Program Description/Abstract Dendritic cells (DCs) are the central regulators of T cell immunity and tolerance. Conventional type 1 or CD8?+ DCs (cDC1) are critical for anti-tumor immunity by presenting antigens to CD8 cytotoxic T cells and stimulating their expansion and effector function within tumors, and cDC1 function is directly associated with immune- mediated tumor rejection and the success of immunotherapies. Therapies aimed at enhancing cDC1 function may therefore boost anti-tumor immunity and potentially increase the responsiveness of cancer patients to immunotherapies such as checkpoint blockade. Although lineage-specific transcriptional regulators of cDC1 development such as Batf3 have been identified, the molecular pathways and cellular processes that selectively orchestrate cDC1 function remain poorly defined. Metabolic reprogramming is important for DC development and activation, but metabolic dependence and regulation of DC subsets are unknown. We have developed a novel data-driven systems biology algorithm (NetBID) to identify ?hidden? drivers in DC subsets, and discovered an unexpected role of Hippo pathway kinases, Mst1 and Mst2 (Mst1/2), in selectively programming cDC1 function and metabolism. The NetBID analysis reveals a marked enrichment of the activity of Hippo pathway kinases in cDC1 relative to conventional type 2 or CD8?? DCs (cDC2). Deletion of Mst1/2 in DCs disrupts CD8 T cell function and anti-tumor immunity. Mst1/2-deficient cDC1 but not cDC2 are impaired in priming CD8 T cells in vivo and in vitro. Mechanistically, cDC1 show much stronger oxidative phosphorylation (OXPHOS) and are critically dependent upon Mst1/2 signaling to maintain bioenergetic activities and mitochondrial dynamics for functional capacities. We therefore hypothesize that the interplay between Hippo/Mst signaling and mitochondrial activity orchestrates a unique metabolic checkpoint in cDC1 to direct CD8 T cell responses and anti-tumor immunity. Aim 1. Establish Mst1/2 signaling in tumor DCs and anti-tumor immunity. Aim 2. Establish mitochondrial function and metabolic control of DC subsets. Aim 3. Determine Hippo pathway and signaling circuits in DC subsets. There has been little description of the molecular pathways or metabolic programs that control the unique function of DC subsets. We predict our studies will establish a new paradigm in DC biology and immunometabolism with the potential to translate into innovative strategies for cancer treatment.